Quantitative Tracking of Isotope Flows in Proteomes of Microbial Communities

Nitrogen or carbon flows from 15N- or 13C-enriched substrates into the biomass of a microbial community can be traced by measuring the incorporation of these stable isotopes into biomarkers such as lipids, nucleic acids, and proteins. This technique, called stable isotope probing (SIP)1, has been used to elucidate metabolic activities in microbial communities (1). The first example of SIP used lipids as the biomarker (2). A low level of 13C incorporation in lipids can be determined by gas chromatography-mass spectrometry, but it is generally difficult to link lipids to specific microorganisms. More commonly, DNAs or RNAs are used as the biomarkers for SIP analysis (3). Isotopically labeled nucleic acids are separated from unlabeled nucleic acids using buoyant density gradient centrifugation. Analysis of the separated nucleic acids provides direct information about which microorganisms have incorporated the label. However, gradient centrifugation can only resolve nucleic acids with large differences in the degree of label incorporation. Recently a protein-based SIP method was developed that uses mass spectrometry (MS) to determine the extent of 13C incorporation of peptides and proteins (4–9). Because the degree of label incorporation in proteins and peptides can be determined to high resolution by mass spectrometry, and as proteins and peptides contain sequence information that links each molecule to its organism of origin, protein-based SIP methods enable determination of low levels of isotope incorporation into microorganisms that can be resolved at the strain level. Early protein SIP studies determined the 13C atom% of up to 38 proteins from a single organism in a pure culture or an enrichment culture. Organisms studied included Methylibium petroleiphilum (8) and Aromatoleum aromaticum (4). In both studies, proteins of unknown 13C atom% were identified by matching their gel spots from two-dimensional gel electrophoresis to spots from unlabeled proteomes. The 13C atom% was estimated using least-square fitting analysis of isotopic distributions of detected peptides. The measured 13C atom% of a limited number of proteins allowed tracking of 13C from a substrate into a species of interest, but this number of proteins is insufficient for intra-organismal, pathway-resolved comparisons of label incorporation dynamics. More recently, a new method based on peptide decimal place slope was developed for protein SIP (7, 8). This method requires aggregation of at least 100 peptides for precise 13C atom% estimation and allows estimation of aggregate 13C atom% in an organism's proteome. However, it cannot resolve individual proteins' atom% or pathway-specific differences. Here, we report a proteomic SIP method that can determine 15N atom% of thousands of identified proteins from multiple strains and species in a model microbial community. Compared with the existing SIP methods, this new proteomic SIP method provides a deep coverage of strain-resolved data sets with variable extents of incorporation of 15N/13C. The approach provided new insights into the metabolic activity of established and regrowing multispecies biofilm communities.